Myotonic dystrophy (dystrophia myotonica, DM) is the most common form of muscular dystrophy in adults that affects 1 in 8500 individuals worldwide. The genetic mutations responsible for DM were identified as the expanding (CUG)n repeats in the 3'UTR of DMPK mRNA (for DM1) or the (CCUG)n expansion in the intron of ZNF9 (for DM2). Such non-coding RNA repeats bind and sequester muscleblind proteins or change the expression of the CUG binding proteins that regulate alternative splicing of endogenous genes critical to muscle and heart function. Currently there is no cure for DM, although complications of the disease, including heart problems and cataracts, can be treated and alleviated. The molecular therapeutic strategy such as conventional gene therapy that restore muscleblind protein or antisense oligos against RNA repeats had produced some promising results. However, conventional gene therapy approaches are limited by the fact that the RNA repeats can positively or negatively affect expressions of multiple splicing factors, therefore restoring one gene cannot efficiently reverse the symptoms. The antisense method, on the other hand, is limited by the difficulties of delivering oligonucleotides into muscle or heart. Here we propose a novel approach to target and cleave the expanding RNA repeat with the artificial site- specific RNA nucleases (ASREs) that were firstly engineered in our lab. Such artificial enzymes were constructed with an RNA binding module (PUF domain of PUM1) that is specifically designed to recognize any 8-nt sequence and an endoribonuclease domain (PIN domain of SMG6) that efficiently cleave RNA. We will design novel ASREs that can specifically bind and cleave expanding RNA repeats in the cell nucleus where the toxic RNAs are accumulated. We will focus on the DM1 that is the most common and severe form of myotonic dystrophy. Specifically, we will first identify the C binding code for PUF domain and engineer new PUF domains that specifically bind to the (CUG)n repeats, and further generate designer ASREs using these PUFs. We will determine if expression of ASRE in DM1 cells can restore the normal expression level and localization pattern of CUG-binding protein 1 (CUGBP1) and muscle blind-like 1 (MBNL1). In addition, we will examine if the ARSE treatment can reverse the mis-splicing of genes affected in patients and transgenic DM1 mouse (such as CIC-1, cTnT and SERCA1). Finally, we will use mRNA-seq to determine how ASRE treatment affects the expression and splicing of all genes in DM1 cells. Such information will help the future application of ASRE in DM1 mouse model and DM1 patients in the next stage of this project. Cumulatively, our studies will establish basis for a novel therapeutic approach for DM treatment. Combined with the AAV vectors that can efficiently deliver genes to human muscle and heart, this unique method will provide an effective treatment that can be tested in DM1 animal model and eventually in DM1 patients. PUBLIC HEALTH RELEVANCE: This proposal is focused on the development of a new therapeutic approach for myotonic dystrophy treatments. Myotonic dystrophy (DM) is a dominantly inherited disorder with multisystemic clinical features affecting skeletal muscle, heart, eyes, and endocrine system. It is the most common form of muscular dystrophy in adults (affecting 1 in ~8500 people worldwide), and is caused by non-coding RNA expansion that affects the level of several important splicing factors. Currently there is no cure for DM, although complications of the disease, including heart problems and cataracts, can be treated and alleviated.